In the first step in insulin action, insulin binds to its receptor on the surface of the target cell, thereby activating the insulin receptor tyrosine kinase. This leads to receptor autophosphorylation, and to phosphorylation of tyrosine residues in multiple intracellular proteins. We have investigated the pathway whereby insulin stimulates the translocation of GLUT4 glucose transporters to the plasma membrane in isolated rat adipocytes, a physiologically important target cell for insulin action. Using an experimental approach involving transient expression in primary cultures of rat adipocytes, we have previously demonstrated that insulin receptor tyrosine kinase activity is required for this action of insulin, that IRS-1 appears to be at least one of the substrates which participates in mediating the effect of insulin upon recruitment of GLUT4, and that activation of phosphatidyl inositol 3-kinase (PI 3-kinase) activity also appears to play a necessary role in mediating this action of insulin. More recently, we have extended these studies in several ways: (1) We have cloned the cDNA encoding murine IRS-3, another member of the insulin receptor substrate family. (2) We have demonstrated that both murine IRS-2 and murine IRS-3 can substitute for IRS-1 in mediating the translocation of GLUT4. (3) We have demonstrated that protein kinase B (also known as Akt) appears to be downstream from PI 3-kinase in the pathway whereby insulin stimulates recruitment of GLUT4 to the plasma membrane.